A total of 18 plaque-morphology mutants of herpes simplex virus type 1 (HSV-1) have been isolated from two parental strains of HSV-1 which, unlike the parents, cause extensive fusion of human embryonic lung (HEL) cells in an otherwise normal infection. The mutants examined can be divided into two complementation groups. Representatives of both groups incorporate reduced amounts of major fully glycosylated viral glycorproteins into plasma membranes of infected cells, and synthesize at least one of these glycoproteins (B2) in progressively reduced amounts as infection proceeds. The putative precursor glycoprotein is synthesized in normal amounts throughout infection. In vitro mutagenesis of isolated DNA restriction fragments will be used to search for mutations that may cause fusion in genes derived from all parts of the HSV-1 genome. Immune cytolysis with antisera directed against the major viral glycoproteins will be used to enrich a mutagenized virus population for syn mutants, or other mutants, that cause decreased incorporation of glycoproteins into plasma membranes. Existing and new syn mutants will be physically mapped by transferring mutations from isolated DNA fragments to intact DNA during transfection. Restriction enzyme analysis of DNA of HSV-1/HSV-2 intertypic recombinants containing the syn HSV-1 mutation will also be employed. Glycoprotein processing will be examined for different mutants and a variety of experimental conditions using a pulse-chase labeling procedure and analysis of glycopeptides on one-dimensional SDS-gel electrophoresis or two-dimensional (2D) electrophoresis. Combining these techniques with enzyme treatment of the cell surface to increase the mobility of glycoprotein B2 will allow the determination of the time of synthesis of fully glycosylated B2 and its movement to the plasma membrane. A pulse-chase labeling protocol will be used to determine if virions initially acquire precursor or fully glycosylated glycoproteins when they acquire a membrane. Finally an in vitro fusion assay will be developed which employs a biological indicator capable of amplifying and detecting a single fusion event between two cells.